The mechanism proposed for the catalytic action of the pancreatic enzyme carboxypeptidase A on esters and peptides involves the postulated intermediacy of anhydrides formed between the carbonyl group of the substrate and the carboxylate group of Glu-270. To test this mechanism, substrates like o-aminophenylacetyl-L-phenylalanine which contain internal nucleophiles will be reacted with the enzyme in the hope that cyclic products might be detected from the attack of the internal nucleophile on the anhydride carbonyl group in the decomposition of the latter species. Additionally, we hope to develop a "reverse burst" procedure employing an inhibitor containing a hydrolytically labile group for the active site titration of carboxypeptidase A solutions. The most important aspect of our work, however, will be concerned with the study of the roles of the metal ions present at the active sites of various metallocarboxypeptidases such as the Co ions, Ni ions, Mn ions, Cd ions, Hg ions and even the Cu ions species. This study will be performed by examining the kinetics of hydrolysis of selected substrates by both the modified and unmodified enzymes. In the case of carboxypeptidase Y, attention will be focused first on the nature of the acyl-enzymes detected in the course of the enzyme's esterase action and on establishing whether similar intermediates are formed in the expeptidase action. From both chemical modification experiments and kinetic studies we intend to develop a reasonable mechanistic picture for the action of this enzyme.